PspA, a surface protein present on all pneumococci, acts as a virulence factor that can elicit protective antibodies in mice. Surface-attached PspA inhibits the clearance of pneumococci from the blood of mice, probably by inhibiting opsonophagocytosis, although the mechanism of action is not known. The genes encoding PspA from several different strains of the pneumococcus have been sequenced; the protein has a conserved choline-binding domain (involved in the attachment of PspA to the pneumococcal surface) and a variable -helical coiled-coil domain with epitopes that elibcit protective antibodies. A proline-rich domain between the other two domains may allow the protein to span the bacterial cell wall. Immunization of mice with a plasmid containing pspA/Rx1 provided protection against otherwise fatal pneumococcal challenge (one of the first reports of successful genetic immunization against an extracellular bacterial pathogen. The proposed studies will identify those fragments of pspA that are expressed most efficiently in eukaryotic cells. A genetic construct expressing the entire -helical domain has already been tested and it elicits protection against pneumococcal challenge. Additional constructs from within the -helical domain will be made and tested to identify the optimal fragment for use in genetic immunization. Cytokines will also be used to direct the immune system to a Th2 antibody-mediated response. These studies should provide information on those cytokines important in eliciting protective immunity against extracellular bacterial pathogens during genetic immunization, information that might prove more generally useful than just to pneumococcal immunization. The PI will also examine the level of cross protection elicited by genetic immunization with pspA.